The present invention describes a new method for synthesis of an amino-deoxy-disaccharide or an amino-deoxy-oligosaccharide and N-containing saccharides.
It has been found that the oligosaccharide part of various glycoconjugates (especially glycolipids and glycoproteins) have a number of important functions in vivo (Biology of Carbohydrates, vol. 2, Ginsburg et al., Wiley, New York, 1984; The Glycoconjugates, vol. I-V, Academic Press, New York; S. Hakomori, Ann. Rev. Biochem., vol 50, pp. 733-64; Feizi, Nature, pp 314, 1985; S. Hakomori, Chemistry and Physics of Lipids, vol. 42, pages 209-33). Among other thing it was found that
the carbohydrate structures are important for the stability, activity, localization, immunogenicity and degradation of glycoproteins;
carbohydrates are antigenic determinants (for example blood group antigens);
carbohydrates function as receptors when bound to cell surfaces for pathogens, proteins, hormones, toxins and during cell-cell interactions;
carbohydrates are important for oncogenesis, since specific oligosaccharides have been found to be cancer-associated antigenic determinants;
frequently, only a smaller sequence (di- or trisaccharide) of the carbohydrate part of the glycoconjugate is required for full biological activity (e.g. receptor activity).
Universities and industry are at present working intensely on developing the use of biologically active oligosaccharides within a number of different fields, such as
novel diagnostics and blood typing reagents;
highly specific materials for affinity chromatography;
cell specific agglutination reagents;
targetting of drugs;
monoclonal antibodies, specific against e.g. cancer-associated reagents;
therapy;
development of a new type of therapy, as an alternative to antibiotics, based on the inhibition of the attachment of bacteria and virus on cell surfaces with specific oligosaccharides;
stimulation of the growth of plants and protection against pathogens.
Besides the above mentioned areas, a considerable future market is envisaged for fine chemicals based on biologically active carbohydrates.
Amino-saccharides, where an --OH group in the saccharide is exchanged for an --NH.sub.2 group, in several cases have a higher (or modified) biological activity than the corresponding hydroxyl- or N-acetylamino-deoxy-saccharides, e.g. in the binding to selectins important for the initiation of inflammation processes (binding of leucocytes to epithelial cells in blood vessels). The opportunity to use such saccharides therapeutically, e.g. in acute or chronic inflammatory conditions (e.g. reperfusion, injury, and septic shock) is investigated. An important component in this and in other cases is the selective synthesis of di- and oligosaccharides in sufficient quantities. The present invention describes novel techniques for synthesis of amino-saccharides and novel techniques for synthesis N-containing saccharides from such amino-saccharides.
Amino-deoxy-di-, tri- or higher oligosaccharides which contain one or more amino --NH.sub.2 groups are of high interest for food, agricultural, pharmaceutical or diagnostic applications of carbohydrates, to modify the metabolism of the substance and/or to increase the biological effect of the natural substance.
About ten different monosaccharides are included in the carbohydrate part of the glycoconjugates: D-glucose (Glc), D-galactose (Gal), N- acetyl-D-glucosamine (GlcNAc), N-acetyl-D-neuraminic acid (Neu5Ac), D-mannose (Man), L-fucose (Fuc), N-acetyl-D-galactosamine (GalNAc), xylose (Xyl), and arabinose (Ara) (the abbreviations in brackets are according to IUPAC-IUB's abridged terminology for monosaccharides, J.Biol.Chem. (1982), vol. 257, pages 3347-3354, in which publication one also can find the nomenclature used in this text to describe oligosaccharide sequences). The number of possible structures will be almost infinitely great because both the anomeric configuration and the position of the O-glycosidic bond can be varied.
The organic chemical techniques used today for synthesis of these oligosaccharide structures require an extensive protective group chemistry with many steps of synthesis and expensive catalysts (see e.g. Binkley: Modern Carbohydrate Chemistry, Marcel Dekker, New York, 1988, with references). Low total yields are obtained in these complicated reaction schemes and the technique is not favorable, especially for larger scale; work.
Selective chemical synthesis of amino group containing carbohydrates and derivatives require advanced protection group chemistry with many synthetic steps. (see e.g. Binkley: Modern Carbohydrate Chemistry, Marcel Dekker, New York, 1988, with references). Efficient techniques for preparation of such carbohydrates and derivatives thereof are thus desired.
The present invention describes a process which makes possible a drastically simplified synthesis of derivatised or unmodified di-, tri-, and higher oligosaccharides which contain at least one --NH.sub.2 (amino) group, and a process for the synthesis of N-containing saccharides from such derivatised or unmodified di-, tri-, and higher oligosaccharides which contain at least one --NH.sub.2 (amino) group. Carbohydrate amino derivatives which required several reaction steps to synthesis with previous methods, can, with the method according to the present invention, now be obtained with only one reaction step and with absolute stereo specificity.
Enzymes are nature's own catalysts with many attractive characteristics, such as higher stereo-, regio-, and substrate selectivity as well as high catalytic activity under mild conditions. Today, great hopes are therefore placed in being able to utilize enzymes for large-scale selective synthesis of oligosaccharides with fewer reaction steps and consequently higher total yields than by organic chemical methodology.
Both hydrolases (glycosidases, EC 3.2) and glycosyltranferases (EC 2.4) can be used for synthesis (glycosidases: see Nisizawa et al, in "The Carbohydrates, Chemistry and Biochemistry", 2nd Ed., vol. IIA, pages 242-290, Academic Press, New York, 1970). With glycosidases, reversed hydrolysis (equilibrium reaction) or tranglycosylation (kinetic reaction) are often used to obtain synthesis (see e.g. K.G.I. Nilsson, Carbohydr. Res. (1987), vol. 167, pages 95-103; Trends in Biochemistry (1988), vol. 6, pages 256-264). ##STR1## (DOH is donor saccharide, DOR is donor glycoside with .alpha.- or .beta.-glycosidically bound aglycon (--R), HOA is acceptor saccharide and EH is enzyme).
With transferases, a nucleotide sugar (non-limiting examples are UDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc, etc), which is relatively expensive, is used as donor. Furthermore, glycosidases are abundant and can often be used directly without purification.
The synthetic method according to the invention includes at least one process characterized by that a glycosidase (EC 3.2) is used to catalyze an equilibrium or a transglycosylation reaction between an acceptor substance, which consists of a mono-, di-, tri- or higher oligosaccharide which contains at least one amino-deoxy-group ##STR2## and which is modified or unmodified, and a glycosyl donor, which is a monsaccharide, disaccharide, oligosaccharide or a glycoside or derivative thereof, and that the product is used for continued synthesis and/or is isolated from the product mixture.
In this way one obtains, according to the invention, stereospecific synthesis of di-, tri-, or higher amino-deoxy-oligosaccharides or derivatives thereof, which can be used directly, or after further synthesis, for a number of various applications, e.g. for pharmaceutical/medical/diagnostical studies, for applications in therapy or diagnostics, as additives in cosmetics or in food, for modification of separation material, affinity chromatography, modification of amino acids, peptides, proteins, fatty acids, lipids, enzymes, or recombinant proteins.
In the synthesis according to the invention, the capacity of glycosidases to form stereospecific glycosidic linkages between a glycosyl donor (DR in the scheme below, where D symbolizes the transferred carbohydrate part) and a glycosyl acceptor (HOA), summarized in the scheme below: ##STR3## The reaction according to the invention can be carried out according to two principles, either with equilibrium controlled synthesis (R=H), or with transglycosylation reaction (R=F, or an organic group; kinetically controlled reaction). These general types of reactions are well know to the expert and their carrying out, as well as the choice of glycosyl donor and glycosidase, do not restrict the scope of the invention.
Nitrogen-containing saccharides, e.g. of the type illustrated in the figures below are of interest in several connections. ##STR4## in which I symbolizes a derivatized 2-amino-2-deoxy-D-glucopyranoside (derivatized GlcN) and II symbolizes a derivatized 2-amino-2-deoxy-D-galactopyranoside (derivatized GalN) and R symbolizes hydroxyl groups or organic or inorganic groups (e.g. --NR.sup.2 =NH.sub.2 or --NHAc group) and in which at least one of R.sup.1, R.sup.3, R.sup.4 or R.sup.6 is constituted by a mono-, di-, tri- or higher oligosaccharide group which is glycosidically bound to I or II and is, for the rest, non-derivatized or is derivatized with one or more organic or inorganic groups (as defined above). Examples of such saccharides are Lewis-a, Gal.beta.1-3(Fuc.alpha.1-4)GlcNAc and Lewis-x blood-group structures and derivatives thereof and of other biologically active oligosaccharides in which the oligosaccharide derivative is defined here and below in that the saccharides are substituted as Lewis-a or Lewis-x in at least one hydroxyl group and/or in amino-deoxy position with an organic (e.g. an aliphatic, aromatic group or a saccharide group) or inorganic group (sulfate, carboxyl, phosphate group, for example).
Examples of other saccharides are saccharides containing at least one of an .alpha.- or .beta.-glycosidically linked sialyl-, D-xylosyl-, D-mannosyl-, N-acetyl-D-glucosaminyl-, N-acetyl-D-galactosaminyl- or D-glucosyl- group.
These and other derivatized 2-amino-2-deoxy-saccharides (derivatized ManN) have several interesting biological applications.